Synapses are fundamental to neuronal communication. As most clearly demonstrated in the CA1 field of rodent hippocampus, synaptic efficacy can vary over time. This synaptic modulation underlies many aspects of higher brain function. Its dysfunction is implicated in developmental disorders and certain types of epilepsy, and may be important for a variety of neuropsychiatric diseases. It is now generally agreed that proteins lying within the postsynaptic density (PSD) play a key role in regulating synaptic efficacy, but the organization of these proteins within the PSD remains largely unknown. Elucidating the supramolecular architecture of the PSD is the long-term goal of this project. Current evidence suggests that each NMDA receptor combines with numerous signaling, adaptor, and cytoskeletal proteins to form individual semi-autonomous signaling "modules," but these are now understood only as biochemical abstractions. The proposed research combines immunogold EM with high-resolution electron tomography to study the NMDAR module as an organized physical structure, determining its size and shape, and examining its internal organization. It also explores the organization of these modules into larger domains within the PSD, and investigates the relationship of the PSD to the cytoskeleton. Specific Aim 1 determines the morphology of NMDAR signaling modules; Specific Aim 2 elucidates the internal organization of NMDAR modules, using immunogold mapping to characterize the laminar organization of four major proteins within a module; Specific Aim 3 examines the distribution of NMDAR modules along the synaptic apposition; and Specific Aim 4 investigates contacts between the PSD and the actin cytoskeleton, and the organization of actin filaments within the spine. This research proposal addresses the physical organization of molecular signaling pathways at synapses in the hippocampus, focusing on proteins associated with an especially important type of neurotransmitter receptor, the NMDA receptor. Successful completion of this work will improve our understanding of synaptic mechanisms in the brain, will provide new clues regarding fundamental processes that underlie learning and memory, and may help understand the causes of a variety of neurological, developmental, and psychiatric disorders.